Temporal changes in expression of FoxA1 and Wnt7A in isolated adult human alveolar epithelial cells enhanced by heparin.

Pre- and postnatal developmental studies of the lung have provided compelling evidence demonstrating multiple factors that orchestrate alveolar epithelial cell differentiation. The extent to which reactivation of certain developmental pathways in the adult might influence the course of differentiation of alveolar type 2 cells (AT2) into AT1 cells is not known. In this study, we examined selected members of the forkhead (Fox) family of transcription factors and the Wnt (wingless) family of signaling proteins for expression during human alveolar cell differentiation in vitro and determined their potential responses to sulfated components of extracellular matrix (ECM), like those shed from cell surfaces or found in basement membrane and modeled by heparin. Isolated adult human AT2 cells cultured over a 9-day period were used to define the temporal profile of expression of targeted factors during spontaneous differentiation to AT1-like cells. FoxA1 protein was upregulated at early to intermediate time points, where it was strongly elevated by heparin. Gene expression of wnt7A increased dramatically beginning on day 3 and was enhanced even further on days 7 and 9 by heparin, whereas protein expression appeared at days 7 and 9. These temporal changes of expression suggest that sulfated ECMs may act to enhance the increase in FoxA1 at the critical juncture when AT2 cells commence the differentiation process to AT1 cells, in addition to enhancing the increase in wnt7A when the AT1 cell phenotype stabilizes. Collectively, these factors may act to modulate differentiation in the adult human pulmonary alveolus.

Alterations in cytoskeletal and immune function-related proteome profiles in whole rat lung following intratracheal instillation of heparin.

BACKGROUND: Heparin has been shown to modify fundamental biologic processes ranging from blood coagulation and cell proliferation to fibrogenesis and asthma. The goal of this study was to identify specific or broad biologic responses of the rat lung to intratracheal instillation of heparin by targeted proteomic analysis. METHODS: Rats were given either aerosolized 500 microg heparin in 250 microl saline or saline alone. Lungs were harvested at 0, 24, or 96 hours post-treatment and isolated proteins analyzed by two-dimensional gel electrophoresis. Proteins which increased and decreased significantly in treated groups above controls were then selected for identification by mass spectrometry. RESULTS: Although heparin treatments resulted in a general reduction in cytosolic protein expression, there were significant increases within members of discrete groups of proteins. At 24 hours, proteins which function in cytoskeletal organization and in calcium signaling were up-regulated between 2- and 27-fold above baseline and untreated controls. Increased proteins include annexins V and VI, septin 2, capping G protein, actin-related protein 3, moesin, RhoGDP dissociation inhibitor, and calcyclin. A group of proteins relating to immune response and tumor suppressor function were either up-regulated (tumor suppressor p30/hyaluronic acid binding protein-1, Parkinson disease protein 7, proteosome 28 subunit/interferon-gamma inducible protein, and proteosome subunit macropain alpha-1) or strongly down-regulated (transgelin). At 96 hours, most proteins that had increased at 24 hours remained elevated but to a much lesser degree. CONCLUSION: These cumulative observations demonstrate that whole lung heparin treatment results in significant up-regulation of selected groups of proteins, primarily those related to cytoskeletal reorganization and immune function, which may prove to be relevant biomarkers useful in analysis of lung exposures/treatments as well as in system biology studies.

Heparin and fibroblast growth factors affect surfactant protein gene expression in type II cells.

The stimulation and maintenance of the pulmonary alveolar type II cell's capacity to biosynthesize, store, and secrete surfactant proteins (SPs) are modulated to a great extent by growth factors, extracellular matrix (ECM) components, and hormones. It is possible that differences in ECM composition, as exist between type I and II cells normally or as might occur with excessive cell surface shedding during inflammation or injury states, may specifically alter SP expression. Here, isolated type II cells were exposed to the model sulfated ECM heparin; desulfated heparin; and/or fibroblast growth factor (FGF)-1, -2, or -7 for 24 h to examine by quantitative real-time polymerase chain reaction their effects on SP gene expression. Aquaporin 5 (AQP-5) gene expression was also examined as a phenotypic marker for the type I cell. SP-B mRNA abundance was increased 4- to 8-fold by all three FGFs. Heparin at low concentrations (5 microg/ml) or desulfated heparin at high concentrations (500 microg/ml) enhanced the effects of FGF-2 and -7, while high heparin concentrations (500 microg/ml) were inhibitory. In contrast, SP-B mRNA abundance was increased by heparin in a dose- and sulfation-dependent manner when used in combination with FGF-1. SP-C and AQP-5 mRNA levels were increased by heparin alone in a dose- and sulfation-dependent manner, while all FGFs lacked effect on SP-C or AQP-5 mRNA levels. These data indicate that heparin can be stimulatory to SP gene expression depending on concentration, degree of sulfation, and surrounding FGF environment, and that heparin plays a significant role in modulating alveolar epithelial cell phenotype in vitro.

Heparin affects signaling pathways stimulated by fibroblast growth factor-1 and -2 in type II cells.

Undersulfation of the basement membrane matrix of alveolar type II (AT2) cells compared with that of neighboring type I cells is believed to account for some of the known morphological and functional differences between these pneumocytes. Heparin, a model for sulfated components of basement membrane matrices, is known to inhibit fibroblast growth factor (FGF)-2-stimulated DNA synthesis as well as gene expression of FGF-2 and its receptor in AT2 cells. To determine whether these end points result from specific effects of heparin on FGF-related signaling pathways, isolated rat AT2 cells were treated with 100 ng/ml FGF-1 or FGF-2 in the presence of up to 500 microg/ml heparin. In addition, experiments were done on cells grown in the presence of 20 mM sodium chlorate (sulfation inhibitor). High-dose heparin reduced FGF-1- or FGF-2-stimulated phosphorylation of mitogen-activated protein kinase kinases (MEK1/2), p44/42 mitogen-activated protein kinases (MAPK/ERK1/2), stress-activated protein kinase/c-Jun NH(2)-terminal kinase, Akt/protein kinase B, and p90(RSK). FGF-2-stimulated signaling was more sensitive to heparin's effects than was signaling stimulated by FGF-1. Heparin had an additive effect on the reduced [(3)H]thymidine incorporation in FGF-2-treated AT2 cells caused by inhibition of the MEK/ERK pathway by the MEK inhibitor PD-98059. The data suggest that heparin's known capacity to alter DNA synthesis and, possibly, other biological end points is realized via cross talk between multiple signaling pathways.

Heparin inhibits DNA synthesis and gene expression in alveolar type II cells.

Responses of isolated type II alveolar cells to fibroblast growth factors (FGF) have been shown to be sensitive to the level of sulfation in extracellular matrix (ECM) substrata. These observations may reflect the specific in situ distribution and level of sulfation of ECM within the alveolar basement membranes (ABM) associated with type II cells. The goal of this study was to determine if the model sulfated ECM heparin modified DNA synthesis and gene expression by type II cells in a concentration dependent-manner. Isolated rat type II cells were exposed to different concentrations of heparin (0.005-500 micro g/ml) in serum-free medium for 1-3 d with or without FGF-1 or FGF-2. The effects of heparin were examined by [(3)H]thymidine incorporation into DNA, total cell protein, cell number, and selected gene expression. Results indicated that heparin inhibited [(3)H]thymidine uptake in a concentration-dependent manner. Total protein, cell number, and FGF-2 protein expression and mRNA of FGF-1, -2, and FGF receptor-2 detected by reverse transcriptase-polymerase chain reaction were decreased by heparin. These results demonstrate that sulfated molecules in the ABM may play important regulatory role(s) in selected type II cell activities during normal cell homeostasis, turnover, and repair after lung injury.

Effect of growth factor-fibronectin matrix interaction on rat type II cell adhesion and DNA synthesis.

Type II cells attach, migrate, and proliferate on a provisional fibronectin-rich matrix during alveolar wall repair after lung injury. The combination of cell-substratum interactions via integrin receptors and exposure to local growth factors are likely to initiate the signals required for cell proliferation, differentiation, reepithelialization, and ultimate restoration of the alveolar wall structure. Accordingly, primary cultured type II cells have been shown to bind fibronectin, in part through the alpha5beta1 integrin, and to respond to growth factors that induce type II cell proliferation, such as fibroblast growth factor 1 (FGF-1). The purpose of this study was to determine whether or not FGF-1 modifies type II cell attachment to fibronectin, and if together they affect DNA synthesis. Attachment assays showed that FGF-1 treatment enhanced type II cell adhesion to fibronectin. This effect correlated with an increase in beta1 integrin cell surface expression, and with the formation of cytoskeletal stabilizing structures such as lamellipodial extensions and stress fibers. FGF-1 also induced an increase in thymidine incorporation into DNA. Together FGF-1 and fibronectin appear to promote adhesion, cytoskeletal organization, and increased DNA synthesis, and in this way influence cell-substratum interactions and signaling during alveolar repair.

Detection of chondroitin sulfates and decorin in developing fetal and neonatal rat lung.

Chondroitin sulfates and their related proteoglycans are components of extracellular matrix that act as key determinants of growth and differentiation characteristics of developing lungs. Changes in their immunohistochemical distribution during progressive organ maturation were examined with monospecific antibodies to chondroitin sulfate, a nonbasement membrane chondroitin sulfate proteoglycan, and the specific chondroitin sulfate-containing proteoglycan decorin in whole fetuses and lungs from newborn and adult rats. Alveolar and airway extracellular matrix immunostained heavily in the prenatal rat for both chondroitin sulfate and chondroitin sulfate proteoglycan, whereas decorin was confined to developing airways and vessels. These sites retained their respective levels of reactivity with all antibodies through 1-10 days postnatal but thereafter became progressively more diminished and focal in alveolar regions. The heavy staining seen early in development was interpreted to reflect a significant and wide distribution of chondroitin sulfates, chondroitin sulfate proteoglycans, and decorin in rapidly growing tissues, whereas the reduced and more focal reactivity observed at later time points coincided with known focal patterns of localization of fibrillar elements of the extracellular matrix and a more differentiated state.